Highly purified antiendotoxin compound

ABSTRACT

The invention provides compositions containing a highly purified antiendotoxin compound and methods of preparing and using such compositions.

BACKGROUND OF THE INVENTION

This invention relates to compositions containing a highly purifiedantiendotoxin compound and methods of preparing and using suchcompositions.

Since the 1930's, the increasing use of immunosuppressive therapy andinvasive devices, as well as the increased incidence of antibioticresistance in bacteria, have led to a gradual rise in the occurrence ofsepsis and septic shock. Currently, the estimated incidences in the U.S.of sepsis and septic shock are 400,000 and 200,000 patients/year,respectively. This results in about 100,000 fatalities/year, makingseptic shock the most common non-coronary cause of death in hospitalIntensive Care Units (ICUs). Currently, ICU therapy for septic shock islimited to antibiotic therapy, cardiovascular resuscitation,vasopressor/ionotrope therapy, and ventilatory support. This ICU carecan cost up to $1,500/day and average a total of $13,000 to $30,000 perpatient. Clearly, any therapy that can reduce the morbidity and cost forthe care of sepsis/septic shock will be of great value.

It is likely that antibiotics themselves can worsen morbidity associatedwith sepsis; their bactericidal action can result in the release ofendotoxin from gram negative bacteria, which is believed to induce manypathophysiological events such as fever, shock, disseminatedintravascular coagulation (DIC), and hypotension. Consequently,medicines for the treatment of gram negative sepsis have been desiredfor some time, especially drugs capable of blocking endotoxin orcytokines derived from endotoxin-mediated cellular stimulation. To thisend, various strategies for treatment have included administration ofantibodies or other agents against lipopolysaccharide (LPS) orcytokines, such as TNF-α and interleukin-1. For various reasons, theseapproaches have failed.

While endotoxin itself exists in multiple molecular forms, theexpression of many of the toxic properties of endotoxin is attributed tothe highly conserved hydrophobic lipid A portion. An effective drug thatacts as an antagonist to this conserved structure is known as E5564(also known as compound B1287 and SGEA). This drug is described ascompound 1 in U.S. Pat. No. 5,681,824, which is incorporated herein byreference. E5564 has the formula: (α-D-Glucopyranose,3-0-decyl-2-deoxy-6-O-[2-deoxy-3-O-[(3R)-3-methoxydecyl)-6-O-methyl-2-[[(11Z)-1-oxo-11-octadecenyl)amino]-4-O-phosphono-β-D-glucopyranosyl]-2-[(1,3-dioxotetradecyl)amino]-,1-(dihydrogenphosphate), which can be provided as a tetrasodium salt. E5564 has amolecular weight of 1401.6.

SUMMARY OF THE INVENTION

The invention provides a composition including a compound, E5564, havingthe formula:

or a pharmaceutically acceptable salt thereof. This composition contains≦0.1%, by weight, of any individual monosaccharide, such as a right orleft side monosaccharide (as shown in formula set forth above), or≦0.1%, by weight, of total monosaccharides.

The invention also includes a composition including E5564, or apharmaceutically acceptable salt thereof, and ≦0.1%, by weight, of anyindividual dibutylphthalate, or ≦0.1%, by weight, of totaldibutylphthalates.

An additional composition that is included in the invention containsE5564, or a pharmaceutically acceptable salt thereof, and ≦0.1%, byweight, of any individual disaccharide contaminant, or ≦0.1%, by weight,of total disaccharide contaminants. One or more of these disaccharidecontaminants can include, for example, a modified β-ketoamide sidechain, or can lack β-ketoamide side chain.

The invention further includes a composition containing E5564, or apharmaceutically acceptable salt thereof; and ≦0.1%, by weight, of anyparticular impurity or all impurities. All of the compositions describedherein can include a pharmaceutically acceptable carrier or diluent.

Also included in the invention are methods of obtaining compositionscontaining compound E5564, or a pharmaceutically acceptable saltthereof, and ≦0.1%, by weight, of a contaminant (e.g., the contaminantsmentioned above). This method involves obtaining a preparation of thecompound and subjecting the preparation to (i) anion exchangechromatography, (ii) preparative liquid chromatography, and (iii) solidphase extraction.

The invention also includes a method of preventing or treatingendotoxemia in a patient by administering to the patient any of thecompositions described herein, which include a pharmaceuticallyacceptable carrier or diluent. Patients that can be treated using themethods and compositions of the invention include, for example, surgicalpatients (e.g., cardiac surgical patients), if appropriate, patientsthat have or are at risk of developing endotoxemia, sepsis, or septicshock, patients that are infected with HIV, and patients who areimmunocompromised due to their suffering from an immunological disorder.The invention further includes the use of the compositions describedherein for preventing or treating these conditions, as well as for thepreparation of medicaments for preventing or treating these conditions.

The invention provides several advantages. For example, the compositionsand methods of the invention provide significant therapeutic benefitsand the methods are easily carried out, especially with many of thepatients treated according to the invention, who already may haveintravenous lines inserted, as part of their treatment in the ICU. Also,because the compositions of the invention are highly pure, they enablethe administration of lower amounts of drug preparation to patients thanwould be possible with a less pure preparation, to yield the sameeffect.

Other advantages of the invention will be apparent from the followingdetailed description, the drawings, and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an HPLC chromatogram, full scale, of a preparation of compoundE5564 prior to purification using the methods of the invention.

FIG. 2 is an HPLC chromatogram, expanded view, of a preparation ofcompound E5564 prior to purification using the methods of the invention.

FIG. 3 is a schematic representation of compounds that may be present inthe compositions of the invention.

DETAILED DESCRIPTION

The invention provides compositions that include a purifiedantiendotoxin compound and methods of preparing and using suchcompositions. In particular, the compositions of the invention includecompound E5564, which has the formula:

or a pharmaceutically acceptable salt thereof, and ≦0.1%, by weight, ofany particular type of contaminant, such as a monosaccharide (e.g., aright or left side monosaccharide), a dibutylphthalate, or adisaccharide (e.g., a disaccharide that includes a modified β-ketoaraideside chain or that lacks a β-ketoamide side chain). Alternatively, thecompositions of the invention can include ≦0.1%, by weight, of anyindividual contaminant.

The compositions of the invention can be prepared using purificationmethods that are described in further detail below. Briefly, compoundE5564 that has been obtained using, for example, the synthetic methodsdescribed in U.S. Pat. No. 5,681,824, is subjected to furtherpurification steps, including anion exchange chromatography, preparativeliquid chromatography, and solid phase extraction. Preferably, all threeof these steps are carried out, in the order set forth above. However,as is understood in the art, these steps and their order can be varied,as long as compositions as described herein are obtained. In addition,as can be determined by those of skill in this art, any of these stepscan be carried out at appropriate points during synthesis of the drug,rather than after synthesis, as is described below.

Purification of E5564 by Ion Exchange Chromatography, LiquidChromatography, and Solid Phase Extraction Using Polystyrene-BasedStationary Phases

As is noted above, compound E5564 can be obtained using the methoddescribed in U.S. Pat. No. 5,681,824. Fractionation of drug obtainedusing this method revealed that the preparation includes material inaddition to the drug (FIGS. 1 and 2). In particular, a column having thefollowing characteristics was used to carry out HPLC analysis of thedrug: YMC ODS AP303, 4.6×250 mm, 5 μm particle size, 300 Å pore size.The injection volume was 25 μL, and the column was run at a flow rate of0.8 mL/minute, at ambient temperature, and monitored at 254 nm UVabsorbance. The mobile phases were A: 20/900/100 H₃PO₄/H₂O/MeOH, and B:20/980 H₃PO₄/MeOH. The gradient used was 10% B: 0-10 minutes, 10-100% B:10-70 minutes, and 100% B: 70-100 minutes. Structures of compounds thatmay be present in the drug preparation are shown in FIG. 3.

To purify the drug further, the crude reaction product, fromconcentration of the last chemical reaction, is dissolved in methanolcontaining at least 5% water. Typically, 4.5 kg of methanol/water isrequired to dissolve 2.9 kg of crude reaction concentrate. This solutionis loaded onto a previously prepared column packed with apolystyrene-based, tetraalkyl ammonium-functionalized ion exchangeresin, such as Source 30Q from Pharmacia Biotech. The size of the idealload can be determined empirically or can be calculated from the loadcapacity of the resin and the moles of exchangeable functionality in thecrude product solution. Initial purification begins by elution ofnon-charged reagents, impurities, and reaction by-products usingmethanol/water mixtures as a mobile phase. Typically, methanolcontaining at least 5% water is an effective mobile phase. The elutionof non-product compounds is typically monitored using an in-line UV/visdetector. After removal of the non-charged species, E5564 is eluted andseparated from the remaining charged impurities by using a mobile phasewith an increasing salt gradient. The nature of the product salt formcan be changed by varying the cation component of this salt gradient.Typically, a slow gradient from 95:5 (v/v) methanol:water to 95:5 (v/v)methanol:4.6 M NaOH is effective for initial purification of E5564 asthe tetrasodium salt. Use of ammonium chloride for the salt gradientallows purification of E5564 as the tetraammonium salt. The separationand purification at this stage is easily monitored by in-line UV/visdetection and/or in-line conductivity detection. Individual fractionscontaining either E5564 or the various by-products are collectedseparately and can be held for isolation and/or further processing.

The product solution from the ion exchange initial purification isanalyzed for product content and quality, and then is used directly as afeed solution for final purification using LC over a non-functionalized,polystyrenedivinylbenzene resin as the stationary phase, such as Source15 RPC from Pharmacia Biotech. A portion of the product-containingsolution from the ion exchange chromatography containing up to 60 g ofE5564 free acid equivalent is loaded onto a previously prepared columncontaining 20-25 L of stationary phase. Purification is effected byelution with mobile phases containing methanol and water or methanol,water, and sodium hydroxide. Typically, the mobile phase uses agradient, but may have extended isocratic portions. The exactcomposition is dependent upon the separation being effected. Forexample, a typical purification mobile phase is isocratic 95:5 (v/v)methanol:0.1 M NaOH for 20 minutes, followed by a gradient to 100%methanol over 15 minutes. Elution of the by-products and E5564 ismonitored by using an in-line UV/vis detector. Fractions containingeither by-products or pure E5564 are collected separately and can beisolated individually, combined with equivalent fractions fromsubsequent injections, or can be held for further processing.

Combined fractions of either pure E5564 or purified by-productscontaining up to 150 g free acid equivalent are typically diluted withwater to a final solvent composition of 50:50 (v/v) methanol:water. Thissolution is loaded onto a column containing 20-25 L of a previouslyprepared polystyrenedivinylbenzene, non-functional resin, such as Source15 RPC from Pharmacia Biotech, as a stationary phase. After loading, themobile phase is changed gradually, through a gradient, from 50:50methanol:water to 100% acetonitrile. During this step, no compoundselute from the column, but water is removed from the system. Afterelution with at least 1 column volume of acetonitrile, the mobile phaseis changed through, preferably, a very steep gradient to 100% methanol.The purified compound elutes in a very concentrated band during thisstep. Elution of the purified compound is monitored by an in-line UV/visdetector. The resultant pure product solution has been concentrated byup to 10 times and the solution is now water free. This improvessubsequent processing substantially. E5564, impurities, and reactionby-products can be purified effectively by the above-describedtechnique.

To reduce the presence of impurities such as dibutylphthalates, thepurification methods described above and/or the original synthesis canbe carried out rising a limited amount of plastics, as dibutylphthalatesare relatively ubiquitous plasticizers.

Use

As is noted above, the compositions of the invention can be used inmethods for preventing and treating diseases and conditions associatedwith exposure to endotoxin. For use in these methods, the compositionsof the invention are formulated to include pharmaceutically acceptablecarriers or diluents. Standard methods for preparing and formulatingdrugs are well known in the art and can be used in the invention. Forexample, the drug, which can be stored as a freeze-dried formulation,can be dissolved in physiological saline (which may include 5% glucose).Alternatively, the drug can be formulated by dissolving 35.4 mg of drugsubstance in 52.1 ml 0.01 N NaOH, stirring for one hour at roomtemperature, and diluting into phosphate-buffered lactose. Afteradjusting the pH to 7.3 and diluting the drug to a final concentrationof 0.1 mg/ml, the solution can be filter-sterilized and lyophilized. Anexample of a formulation of antiendotoxin drug product in 10 ml vials isshown below.

TABLE 1 Material amount E5564 10 mg NaH₂PO₄ 4H₂O qs NaOH qs Lactosehydrous 400 mg Na₂HPO₄H₂O 1.8 mg sterile water 4 mlAdditional appropriate formulations can readily be determined by thoseof skill in this art (see, e.g., Remington's Pharmaceutical Sciences(18^(th) edition), ed. A. Gennaro, 1990, Mack Publishing Company,Easton, Pa.).

The compositions and methods of the invention can be used to prevent orto treat any of a large number of diseases and conditions associatedwith septic shock or endotoxemia. For example, the compositions andmethods of the invention can be used in conjunction with any type ofsurgery or medical procedure, when appropriate, that could lead to theoccurrence of endotoxemia or related complications (e.g., sepsis orseptic shock syndrome). As a specific example, the invention can be usedin conjunction with cardiac surgery (e.g., coronary artery bypass graft,cardiopulmonary bypass, and/or valve replacement), transplantation (of,e.g., liver, heart, kidney, or bone marrow), cancer surgery (e.g.,removal of a tumor), or any abdominal surgery (see, e.g.,PCT/US01/01273).

Additional examples of surgical procedures with which the compositionsand methods of the invention can be used, when appropriate, are surgeryfor treating acute pancreatitis, inflammatory bowel disease, placementof a transjugular intrahepatic portosystemic stent shunt, hepaticresection, burn wound revision, and burn wound escharectomy. Theinvention can also be used in conjunction with non-surgical proceduresin which the gastrointestinal tract is compromised. For example, theinvention can be used in association with chemotherapy or radiationtherapy in the treatment of cancer. The compositions and methods of theinvention can also be used in the treatment of conditions associatedwith HIV infection, trauma, or respiratory distress syndrome, as well aswith immunological disorders, such as graft-versus-host disease orallograft rejection. Pulmonary bacterial infection and pulmonarysymptomatic exposure to endotoxin can also be treated using thecompositions and methods of the invention (see, e.g., PCT/US00/02173).

Administration of the compositions of the invention can be carried outusing any of several standard methods including, for example, continuousinfusion, bolus injection, intermittent infusion, inhalation, orcombinations of these methods. For example, a mode of administration isby continuous intravenous infusion. In such an approach, the infusiondosage rate of the drug can be, for example, 0.001-0.5 mg/kg bodyweight/hour, more preferably 0.01-0.2 mg/kg/hour, and most preferably0.03-0.1 mg/kg/hour, infused over the course of; for example, 12-100,60-80, or about 96 hours. The infusion of the drug can, if desired, bepreceded by a bolus injection; preferably, such a bolus injection isgiven at a dosage of 0.001-0.5 mg/kg. Preferably, the total amount ofdrug administered to a patient is 25-600 mg of drug, more preferably35-125 mg, by infusion over a period of 60-100 hours. As activity in thehospital, and particularly the ICU, is often hectic, minor variations inthe time period of infusion of the drugs may occur and are also includedin the invention.

Additional modes of administration of E5564, according to the methods ofthe invention, include bolus or intermittent infusion. For example, thedrug can be administered in a single bolus by intravenous infusionthrough, for example, a central access line or a peripheral venous line,or by direct injection, using a syringe. Such administration may bedesirable if a patient is only at short-term risk for exposure toendotoxin, and thus does not need prolonged persistence of the drug. Forexample, this mode of administration may be desirable in surgicalpatients, if appropriate, such as patients having cardiac surgery, e.g.,coronary artery bypass graft surgery and/or valve replacement surgery.In these patients, a single bolus infusion of, e.g., 0.10-15 mg/hour(e.g., 1-7 mg/hour or 3 mg/hour) of drug can be administered over aperiod of four hours prior to and/or during surgery. (Note that theamount of drug administered is based on an assumed average weight of apatient of 70 kg.) Shorter or longer time periods of administration canbe used, as determined to be appropriate by one of skill in this art.

In cases in which longer-term persistence of active drug is desirable,for example, in the treatment of a condition associated with long-termexposure to endotoxin, such as during infection or sepsis, or inappropriate surgical situations in which it is determined that prolongedtreatment is desirable, intermittent administration can be carried out.In these methods, a loading dose is administered, followed by either (i)a second loading dose and a maintenance dose (or doses), or (ii) amaintenance dose or doses, without a second loading dose, as determinedto be appropriate by one of skill in this art.

The first (or only) loading dose can be administered in a manner similarto that described for the single bolus infusion described above. Thatis, for E5564 administration, 0.10-15 mg/hour (e.g., 3-7 mg/hour or 3mg/hour) of drug can be administered to a patient over a period of fourhours prior to surgery. If a second loading dosage is to be used, it canbe administered about 12 hours after the initial loading dose, and caninvolve infusion of, e.g., 0.10-15 mg/hour (e.g., 1-7 mg/hour or 3mg/hour) of drug over a period of; e.g., about two hours.

To achieve further persistence of active drug, a maintenance dose (ordoses) of drug can be administered, so that levels of active drug aremaintained in the blood of a patient. Maintenance doses can beadministered at levels that are less than the loading dose(s), forexample, at a level that is about ⅙ of the loading dose. Specificamounts to be administered in maintenance doses can be determined by amedical professional, with the goal that drug level is at leastmaintained. Maintenance doses can be administered, for example, forabout 2 hours every 12 hours beginning at hour 24 and continuing at, forexample, hours 36, 48, 60, 72, 84, 96, 108, and 120. Of course,maintenance doses can be stopped at any point during this time frame, asdetermined to be appropriate by a medical professional.

In the case of pulmonary bacterial infection or pulmonary symptomaticexposure to endotoxin, administration of the compositions of theinvention can be effected by means of periodic bolus administration, bycontinuous, metered inhalation, or by a combination of the two. A singledose is administered by inhalation 1 μg-24 mg, for example, 5-150 μg,or, preferably, 10-100 μg of the drug. Of course, recalcitrant diseasemay require administration of relatively high doses, e.g., 5 mg, theappropriate amounts of which can be determined by one of skill in thisart. Appropriate frequency of administration can be determined by one ofskill in this art, and can be, for example, 1-4, for example, 2-3, timeseach day. Preferably, the drug is administered once each day. In thecase of acute administration, treatment is typically carried out forperiods of hours or days, while chronic treatment can be carried out forweeks, months, or even years.

Both chronic and acute administration can employ standard pulmonary drugadministration formulations. Administration by this route offers severaladvantages, for example, rapid onset of action by administering the drugto the desired site of action, at higher local concentrations. Pulmonarydrug formulations are generally categorized as nebulized (see, e.g.,Flament et al., Drug Development and Industrial Pharmacy21(20):2263-2285, 1995) and aerosolized (Sciarra, “Aerosols,” Chapter 92in Remington's Pharmaceutical Sciences, 16^(th) edition (ed. A. Osol),pp. 1614-1628; Malcobnson et al., PSTT 1(9):394-398, 1998, and Newman etal., “Development of New Inhalers for Aerosol Therapy,” in Proceedingsof the Second International Conference on the Pharmaceutical Aerosol,pp. 1-20) formulations.

All patents and publications mentioned herein are incorporated herein byreference. Other embodiments are within the following claims.

1. A composition comprising a compound having the formula:

or a pharmaceutically acceptable salt thereof, said compositioncomprising ≦0.1%, by weight, of any individual monosaccharide.
 2. Thecomposition of claim 1, wherein said monosaccharide is a right sidemonosaccharide.
 3. The composition of claim 1, wherein saidmonosaccharide is a left side monosaccharide.
 4. The composition ofclaim 1, wherein said composition comprises ≦0.1%, by weight, of totalmonosaccharides.
 5. The composition of claim 1, wherein said compositioncomprises ≦0.1%, by weight of any individual dibutylphthalate. 6-10.(canceled)
 11. The composition of claim 1, wherein said compositioncomprising ≦0.1%, by weight, of any particular impurity.
 12. Thecomposition of claim 11, wherein said composition comprises ≦0.1%, byweight, total impurities. 13-17. (canceled)
 18. The composition of claim1, wherein the monosaccharide has the following structure:


19. The composition of claim 1, wherein the monosaccharide has thefollowing structure:


20. The composition of claim 1, wherein the monosaccharide has thefollowing structure:


21. The composition of claim 1, further comprising a pharmaceuticallyacceptable carrier or diluent.